Shc proteins (hereafter referred to as ShcA) represent major substrates of tyrosine phosphorylation by a wide variety of growth factors and cytokines. We have recently described a novel ShcA-like protein, ShcC, which like ShcA contains an NH-terminal phosphotyrosine binding domain (PTB), a central effector region (CH1) and a COOH-terminal Src homology 2 domain (SH2). Both the SH2 and PTB domains of ShcC bind a similar profile of proteins as the comparable regions of ShcA. In an effort to define the functional differences or similarities between ShcA and ShcC, we have further characterized the PTB domain of ShcC. Using a degenerate phosphopeptide library screen, we show that the PTB domain of ShcC preferentially binds the sequence His-hydrophobic-Asn/hydrophobic-Asn-Pro-Ser/Thr-Tyr(P). This sequence is similar to the binding site for the ShcA PTB domain, suggesting that these two proteins may have overlapping specificities. In addition, random mutagenesis of the ShcC PTB domain has identified several amino acids important for PTB function (Gly, Glu, Ala, Gly, and Asp). Mutation of these amino acids dramatically reduces the affinity of the ShcC PTB domain for the activated epidermal growth factor receptor in vitro.